Stress-induced changes in cognitive function and intestinal barrier integrity can be ameliorated by venlafaxine and synbiotic supplementations

Animals and treatments

Biological samples derived from experimental animals with all treatments were obtained in our previous study. All experimental procedures involving animals in this study were approved by the Thammasat University Animal Care and Use Committee in Pathum Thani, Thailand, under Animal Ethics number 003/2020. The detail of the treated animals, including the experimental groups and treatments, were described by Lapmanee et al. (2023a). Briefly, forty-five adult male Wistar rats (Nomura Siam International Company Limited, Bangkok, Thailand) were housed in groups (2–3 rats/cage) to prevent individual stress and fear under 12 h light (245 ± 5 lux)/12 h dark cycle in an ambient temperature of 25 ± 2 °C and a humidity level of 55 ± 5%. Animals were fed ad libitum with standard diet (CP Company Limited, Bangkok, Thailand) and had unrestricted access to water. After 7 days of acclimatization, rats were randomly divided into five groups, each consisting of an equal number of animals (9 rats/group): (i) healthy control group (Con), (ii) stressed group (Str), (iii) stressed+Vlx group (Str+Vlx), (iv) stressed+Syn group (Str+Syn) and (v) stressed+Vlx+Syn group (Str+Vlx+Syn). Stressed rats were induced to exhibit depressive- and memory impairment-like behaviors by being confined in plastic cones to restrict their movement for 2 h each day at 9:00 AM and 11:00 AM and then returned to their home cage for 14 days. Based on the previous studies by Lapmanee et al. (2017, 2023a), this study freshly prepared all the regimens and utilized sterile normal saline to dissolve synbiotics and/or antidepressants; therefore, normal saline served as the vehicle. Con and Str groups were subjected to oral gavage with normal saline vehicle. In Vlx-treated animals, the antidepressant drugs Vlx (Pfizer Ireland Pharmaceuticals, Co. Kildare, Ireland) a dose of 10 mg/kg body weight was administered orally once daily. In Syn-treated animals, Syn (Inter Pharma Public Co., Ltd., Bangkok, Thailand) containing 1.0 × 10¹⁰ CFU of probiotic strains (including Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium longum, Lactobacillus acidophilus, Lactobacillus casei, and Lactococcus lactis) and oligosaccharide in a 2-gram sample as determined by series dilution, was administered orally once a day. Both Vlx and Syn were freshly prepared and administered daily at 3:00 to 4:00 PM for 14 days. If animals displayed signs of illness, lack of food or water intake, excessive body weight loss exceeding 20%, or poor ambulation in their home cage, rats were excluded from the experiments. Fortunately, no such incidents occurred in the present study. Consequently, the final number of rats per group at the conclusion of the study remained consistent at nine rats.

The body weight of all animals was monitored daily, and they were individually housed in metabolic cages for 24 h on a weekly basis for the assessment of food and water intake. Following the induction of stress and the administration of Vlx and Syn, all rats underwent a series of behavioral tests, including the MWM on days 15 to 17 and FST on day 18. Blind conditions of treatment between analyzers were implemented in the behavioral test.

In each group, four rats underwent oral gavage with fluorescein isothiocyanate (FITC) dextran 1 h before blood collection to study intestinal permeability. The rats were ultimately euthanized using an overdose of isoflurane anesthesia, and subsequently, blood and specific target organs, i.e., the brain and ileum, were harvested for analysis. Whole brains were then rapidly extracted, frozen in liquid nitrogen, and stored at −80 °C for GDNF protein studies. For histomorphological analyses, rats underwent transcardiac perfusion with PBS (pH 7.4) and 4% paraformaldehyde (PFA), followed by 24-h PFA fixation. The processed tissues were embedded in paraffin for further studies. Additionally, urine was collected from the bladder to measure corticosterone (CORT) levels. Wet weights of the adrenal glands, spleen, and thymus were harvested and assessed as markers of stress and immune responses, as previously described (Resendez & Rehagen, 2017; Lapmanee et al., 2017, 2023a). Changes in blood glucose, total cholesterol, triglycerides, and total fat pad weight were recorded as indicators of physical changes and metabolic health (Hyvärinen et al., 2022). The experimental design is shown in Fig. 1.

Morris water maze test

The MWM was utilized to assess learning and memory performance, following the method outlined by Lapmanee et al. (2017). The MWM comprises a circular stainless-steel pool, 150 cm in diameter and 60 cm in height, filled with tap water at a controlled temperature of 23 ± 2 °C, rendered opaque by the addition of 250 mL of milk. Surrounding the pool are cues for spatial orientation, such as black/white circles, crosses, and a black grid. Inside the pool, there is a stainless-steel platform, measuring 10 cm in diameter and positioned 30 cm below the water’s surface, serving as the target. In each trial, a rat was gently placed into one of the four quadrants of the maze: North, South, East, or West. Prior to the commencement of the learning trials, each rat was briefly allowed to become acquainted with the platform by being placed on it for 10 s. If a rat moved away from the platform before the 10 s had elapsed, it was gently returned to its home cage. (i) Spatial learning was assessed through measurements of escape latency. The platform’s location remained constant. If a rat could not locate the platform within 60 s, it was gently placed on the platform, and an escape latency of 60 s was recorded. This training extended over three days, during which the rats completed a total of 20 trials: eight trials on the first two days and four trials on the third day, with 5-min breaks between each trial. An increase in escape latency indicated poorer spatial learning. (ii) The probe test occurred on the third day after training sessions, each lasting 1 h. During this test, the platform was removed from the pool. Spending more time in the correct quadrant (where the platform had been located) indicated better spatial memory.

Forced swimming test

Each rat underwent two swimming sessions within 24 h in a cylindrical container (45 cm height, 25 cm diameter) filled with tap water at 25 ± 2 °C to a depth of 35 cm. During the first 15-min session, the rat was assessed for immobility, swimming, and climbing behaviors. The immobility time in the FST, characterized by the minimal movement to keep the head above water while floating, reflects behavioral despair—a symptom of depression. Swimming involved active forepaw movement, including crossing between quadrants and turning. Climbing was defined as upward forepaw movements along the cylinder’s inner wall. An increase in the duration of immobility or a decrease in the duration of swimming or climbing behaviors were indicative of depression-like behaviors in the rats (Lapmanee et al., 2017; Songphaeng et al., 2023).

Body weight gain, food and water intake

Body weights were measured daily. To calculate the daily body weight gain for each rat, the difference between the final body weight (Day 14) and the initial body weight (Day 1) was divided by 14 days. For food and water intake, measurements were recorded weekly during the experiment. Measurements were taken by weighing the food and measuring the water volume before and after a 24-h period in the metabolic cage (model 3700M071-01CS; Tecniplast, Varese, Italy).

Euthanasia

The rats were euthanized through an overdose of anesthesia inhalation. Isoflurane was used for anesthesia in spontaneously breathing rats. Each rat was situated in a plastic container with a top inlet, and a silicon tube providing a mixture of anesthetic and oxygen gas was connected. Anesthesia was induced by inhaling 5% vaporized isoflurane. It is crucial to note that there were no noticeable movements or reactions to pain stimuli observed in the animals before the commencement of specimen collection.

Blood and urinary biochemical assays

Blood was collected via cardiac puncture between 9:00 AM and 12:00 PM, and serum and plasma were subsequently prepared. Serum glucose, total cholesterol, and triglycerides were assayed using standard methods on an automated clinical chemistry analyzer (Fuji Dri-CHEM NX500i, Tokyo, Japan). Plasma was analyzed for FITC-dextran assay. Rat urinary CORT levels were determined through commercial radioimmunoassay provided by Immunodiagnostic Systems Ltd. (Tyne and Wear, UK).

FITC-dextran assay

After 24 h from the last behavioral test, rats were orally administered 70-kDa FITC dextran (Sigma-Aldrich, St. Louis, MO, USA) at a dose of 25 mg/kg, as reported by Xia et al. (2021). Subsequently, 1 h later, blood was collected into anticoagulant tubes treated with EDTA for plasma preparation. Then, blood samples were vertically kept at room temperature for 15 min. For plasma isolation, the upper parts of blood samples were centrifuged at 2,000 × g for 10 min. Plasma was diluted at a 1:5 (v/v) ratio in phosphate-buffered saline (pH 7.4). Fluorescence intensity was spectrophotometrically measured in 96-well plates using an excitation wavelength of 485 nm and an emission wavelength of 528 nm.

Tissues and organ collections

After completing the behavioral tests, animals were anesthetized using an overdose of isoflurane inhalation. Brains and ileum were carefully dissected free of surrounding tissues, and this dissection process was conducted by a single investigator to maintain consistency. Additionally, the adrenal glands, spleen, thymus, total fat pads from the abdominal cavity, and the area representing fat accumulation, including visceral, retroperitoneal, and epididymal regions, were individually dissected and weighed relative to the final body weight using a similar method. Furthermore, the adiposity index was calculated as the sum of total fats divided by the final body weight, multiplied by 100 (Leopoldo et al., 2016).

Hippocampal and ileum histomorphology

Histological examination of sections stained with hematoxylin and eosin (Sigma-Aldrich, St. Louis, MO, USA) revealed characteristic features in the hippocampal dentate gyrus and small intestine ileum. Tissues from the brain and ileum of four rats in each group were harvested and then preserved in 4% PFA. The target tissues were dehydrated using serial ethanol, cleaned in xylene, and embedded in paraffin. Coronal sections, each with a thickness of 5 µm, were then cut using a microtome (Microsystems, Wetzlar, Germany), mounted on glass slides, and stained using the routine hematoxylin and eosin protocol. Cell death was assessed by the presence of pyknotic cells in the dentate gyrus, while inflammatory cell infiltrate was scored in the ileum (Erben et al., 2014; Akang, 2019). Histological slide interpretations were performed through a blind analysis by two pathobiologists. Each observed intestinal injury was assigned a score, and a cumulative score was calculated at the end of each investigation. The scoring system ranged from 0 to 25 points, with zero indicating no signs of injury to the animals and 25 representing maximum injury, such as the loss of villi. Additionally, the number of Peyer’s patches, crucial to mucosal immunity and modulation of intestinal immune and inflammatory responses, was also observed (Jung, Hugot & Barreau, 2010). Examination of slides, photography, and morphometric studies were conducted at the Pathology Information and Learning Center, Department of Pathobiology, Faculty of Science, Mahidol University, Thailand.

Western blot analysis

The brain was carefully extracted from the skull and the ileum was cleaned with sterile PBS. The dissection of the hippocampus followed the protocols outlined by Lapmanee et al. (2023b). Subsequently, the hippocampal and ileum tissues were combined with a lysis buffer containing protease and phosphatase inhibitor cocktails (Sigma, Burlington, MA, USA). The total protein concentration was quantified using the BCA Protein Assay kit (Thermo Scientific Inc., Waltham, MA, USA). Then, the protein samples (50 μg each) were loaded into the wells of 4–20% Mini-PROTEAN TGX Precast Gels or 15% SDS-PAGE gels depending on their molecular weights and subsequently transferred onto PVDF membranes pre-soaked in methanol (Amersham Biosciences, New Jersey, USA). These membranes were then subjected to overnight incubation at 4 °C with either a 1:1,000 dilution of rabbit polyclonal anti-GDNF antibody (catalog no. Cat #PA5-89957; Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), a 1:5,000 dilution of rabbit polyclonal anti-β-actin antibody (catalog no. AB8227; Abcam, Cambridge, UK) or GAPDH antibody (catalog no. Cat #PA1-988; Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). Following thorough washing steps with TBST, the membranes were exposed to a 1:5,000 dilution of goat anti-rabbit IgG H&L (HRP) secondary antibody (catalog no. AB205718; Abcam, Cambridge, UK) at room temperature for 2 h. Protein bands were visualized using an ECL Prime Western blot detection reagent with the image reader Amersham ImageQuant 500. Densitometry analysis was conducted by software (CYTIVA, Marlborough, MA, USA). The GDNF protein expression level in relation to β-actin or GAPDH in the control group was subsequently normalized to a value of 1.

Statistical analysis

The data are reported as means ± standard error of the mean (SEM). The two data sets were compared using an unpaired Student’s t-test. Group differences were evaluated using one-way analysis of variance (ANOVA) with Dunnett’s multiple comparison test. Statistical significance was set at p < 0.05. All statistical analyses and graphical representations were conducted using GraphPad Prism 8 (GraphPad Software Inc., San Diego, CA, USA).

Syn and Vlx supplementation improves physiological and biochemical parameters changes in stressed rats

As summarized in Table 1, immobilization stress significantly reduced body weight gain (p < 0.01) and daily food intake (p < 0.001) in the stressed rats when compared to non-stressed rats. In contrast, stressed rats exhibited increased water intake (p < 0.001), likely to maintain hydration and eliminate waste. The stressed rats had significantly elevated urinary CORT levels (p < 0.0001) and an increased ratio of wet adrenal gland weight to body weight (p < 0.05) when compared to the control group. Although blood glucose and total cholesterol remained unaffected, stressed rats had higher triglyceride levels (p < 0.05) as compared to the control group, indicating fat breakdown due to stress hormones and decreased adiposity index (p < 0.05). Additionally, immobilized stress reduced thymus weight but not spleen weight (p < 0.05), potentially impairing immune function. Thus, immobilization stress triggers complex physiological responses affecting stress hormones, metabolism, appetite regulation, and immune function, underscoring the multi-system impact of stress in animals.

Supplementation with either Syn or Vlx attenuated stress-induced body weight loss and restored food and fluid intake to normal levels in non-stressed rats. Syn treatment significantly reduced urinary CORT levels (p < 0.0001) in stressed rats, similar to the effect of Vlx treatment. This results show that both Syn and Vlx modulates of neurotransmitter activity in body weight regulation during stress. Both Syn alone or the combination of Syn and Vlx treatments significant reduced blood glucose (p < 0.001) and total cholesterol (p < 0.05) in stressed rats, indicating potential improvement in lipid metabolism due to synbiotic supplementation.

Syn and Vlx supplementation attenuates the stress-induce depression- and memory impairment-like behaviors

MWM was performed to evaluate animals’ learning behavior and spatial memory after 14 days of stress by immobilization and treatments. During the training phase (Fig. 1B), stressed rats exhibited extended escape latency compared to the non-stressed rats, particularly on days 2 (p < 0.01) and day 3 (p < 0.001). In contrast, stressed rats treated with Vlx and/or Syn supplements displayed reduced time to reach the platform on days 2 (p < 0.01). All treatments also reduced time spent swimming to the platform on day 3 (p < 0.001). Additionally, Fig. 1C shows that Vlx and the combination of Syn and Vlx treatment increased the time spent swimming toward the target quadrant (p < 0.001), suggesting the potential of Syn and Vlx to improve learning and spatial memory in stressed rats.

In Fig. 1D, stressed rats demonstrated significantly shorter swimming times compared to the control group (p < 0.05), indicating the manifestation of hopelessness behavior in the FST, a recognized symptom of depression. Interestingly, even though stressed rats treated with Vlx exhibited more immobility time than the control rats (p < 0.0001), they still displayed reduced depression symptoms, as demonstrated by an increase in swimming time (Fig. 1E). Both Syn and Vlx supplementation effectively mitigated depression-like behaviors, as indicated by the reduced immobility time in stressed rats (p < 0.001). Importantly, there were no differences in climbing behavior among the groups, suggesting that stress induction and subsequent treatments did not significantly impact motor activity (Fig. 1F). These results suggest that Syn and Vlx supplementation ameliorate stress-induced depression-like behaviors without altering motor activity.

Syn and Vlx supplementation ameliorates stress-induced neuronal cell death in rat hippocampus

Stress induced markedly changes in the histomorphology of the hippocampus, specifically in the dentate gyrus of male rats, contrasting with the control group. A normal granular cell, characterized by a dark-staining nucleus and scanty cytoplasm, is denoted by the black arrow. Pyknotic nuclei, indicating small, condensed, dark-staining nuclei with eosinophilic cytoplasm, were more prevalent in stressed rats, demonstrating a significantly higher percentage of pyknotic cells in the dentate gyrus (p < 0.0001), indicative of increased neural cell death (see Fig. 2F).

As anticipated, Vlx exhibited neurotherapeutic effects against stress-induced neuronal cell death compared to vehicle treatment. Furthermore, the combination of Vlx and Syn attenuated nerve cell death, reducing the pyknotic index percentage compared with stressed vehicle-treated rats (p < 0.05).

Syn and Vlx supplementation improves intestinal permeability and inflammation in the gut of stressed rats

The morphology of the ileum revealed desquamation of the villi and villous edema in the control group. In contrast, the stressed group exhibited a pronounced loss of villi, along with villous edema and extensive layer separation. Inflammatory signs, including the presence of Peyer’s patches, were observed in the vehicle-treated rats, but these signs were not observed in the other treatment groups (Supplemental Figure). As a result, restraint stress significantly elevated the inflammation score in ileum histomorphology compared to the control group (p < 0.0001). Furthermore, variations in villi numbers, desquamation, flattened villi, and edema layer were observed across all interventions. Despite these commonalities, Syn displayed a mitigated ileum inflammatory score compared to the vehicle-treated group (p < 0.05, as shown in Fig. 3F).

The results of the intestinal permeability study (Fig. 3G) indicated that stressed vehicle-treated rats had higher plasma FITC-dextran levels than the control group (p < 0.0001), indicating a leaky gut condition in stressed rats. Significantly, both Vlx and Syn supplementation were more effective in reducing FITC-dextran levels in stressed rats than the vehicle treatment (p < 0.0001). Furthermore, Syn supplementation appears to reduce intestinal inflammation in stressed rats. Vlx may ameliorate stress-induced intestinal hyperpermeability through modulation of 5-HT and NE activity.

Syn and Vlx supplementation enhances the effect of stress on GDNF expression levels in the hippocampus and gut of rats

The possible molecular mechanisms underlying cellular changes in the hippocampus and ileum of stressed rats were examined (Fig. 2). The expression of the GDNF protein significantly increased in stressed rats (p < 0.0495). In Figs. 2G–2H, treatment with Vlx and/or Syn did not alter hippocampal GDNF protein levels compared to controls or the vehicle treatment group. In the ileum (Fig. 3), stress slightly upregulated GDNF protein levels (p = 0.0525), whereas all treatments were trending to restore GDNF protein levels to controls (Figs. 3H and 3I). These findings suggest that stress negatively impacts brain and intestinal health, and both Vlx and Syn may protect against neural cell death in the dentate gyrus of the hippocampus. Additionally, Syn could offer partial protection by modulating GDNF expression, contributing to the restoration of the disrupted gut barrier caused by stress exposure in male rats.